Purification and Characterization of Novel N-Acyl-D-aspartate Amidohydrolase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6

Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) produced N-acyl-D-aspartate amidohydrolase (D-AAase) in the presence of N-acetyl-D-aspartate as an inducer. The enzyme was purified to homogeneity. The enzyme had a molecular mass of 56 kDa and was shown by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) to be a monomer. The isoelectric point was 4.8. The enzyme had maximal activity at pH 7.5 to 8.0 and 50°C, and was stable at pH 8.0 and up to 45°C. N-Formyl (K_m=12.5 mM), N-acetyl (K_m=2.52 mM), N-propionyl (K_m=0.194 mM), N-butyryl (K_m=0.033 mM), and N-glycyl (K_m =1.11 mM) derivatives of D-aspartate were hydrolyzed, but N-carbobenzoyl-D-aspartate, N-acetyl-L-aspartate, and N-acetyl-D-glutamate were not substrates. The enzyme was inhibited by both divalent cations (Hg²⁺, Ni²⁺, Cu²⁺) and thiol reagents (N-ethylmaleimide, iodoacetic acid, dithiothreitol, and p-chloromercuribenzoic acid). The N-terminal amino acid sequence and amino acid composition were analyzed.     

Preparation of Optically Active Allothreonine by Separating from a Diastereoisomeric Mixture with Threonine

A simple procedure is described to obtain D- and L-allothreonine (D- and L-aThr). A mixture of N-acetyl-D-allothreonine (Ac-D-aThr) and N-acetyl-L-threonine (Ac-L-Thr) was converted to a mixture of their ammonium salts and then treated with ethanol to precipitate ammonium N-acetyl-L-threoninate (Ac-L-Thr·NH₃) as the less-soluble diastereoisomeric salt. After separating Ac-L-Thr·NH₃ by filtration, Ac-D-aThr obtained from the filtrate was hydrolyzed in hydrochloric acid to give D-aThr of 80% de, recrystallized from water to give D-aThr of >99% de. L-aThr was obtained from a mixture of the ammonium salts of Ac-L-aThr and Ac-D-Thr in a similar manner.     

L-Tyrosine Production by Analog-resistant Prototrophic Mutants of Glutamic Acid Producing Bacteria

p-Fluorophenylalanine (PFP) and m-fluorophenylalanine were the most effective inhibitors on the growth of Corynebacterium glutamicum ATCC 13032 among the analogs of phenylalanine and tyrosine tested. Their inhibitory effects were released by L-phenylalanine, and slightly by L-tyrosine and L-tryptophan. 3-Aminotyrosine (3AT), p-aminophenylalanine, o-fluorophenylalanine, and β-2-thienylalanine were weak inhibitors.

Resistant mutants of C. glutamicum isolated on the medium containing both PFP and 3AT or PFP and L-tyrosine were found to accumulate both L-tyrosine and L-phenylalanine, while resistant mutants isolated on the medium containing only PFP were found to produce only L-phenylalanine. Resistant mutants from other glutamic acid producing bacteria isolated on the medium containing both PFP and 3AT or both PFP and L-tyrosine were found to accumulate L-tyrosine and L-phenylalanine.     

Structures and Properties of a Diastereoisomeric Molecular Compound of (2S,3S)- and (2R,3S)-N-Acetyl-2-amino-3-methylpentanoic Acids

An X-ray crystal structural analysis revealed that (2S,3S)-N-acetyl-2-amino-3-methylpentanoic acid (N-acetyl-L-isoleucine; Ac-L-Ile) and (2R,3S)-N-acetyl-2-amino-3-methylpentanoic acid (N-acetyl-D-alloisoleucine; Ac-D-aIle) formed a molecular compound containing one Ac-L-Ile molecule and one Ac-D-aIle molecule as an unsymmetrical unit. This molecular compound is packed with strong hydrogen bonds forming homogeneous chains consisting of Ac-L-Ile molecules or Ac-D-aIle molecules and weak hydrogen bonds connecting these homogeneous chains in a fashion similar to that observed for Ac-L-Ile and Ac-D-aIle. Recrystallization of an approximately 1:1 mixture of Ac-L-Ile and Ac-D-aIle from water gave an equimolar molecular compound due to its lower solubility than that of Ac-D-aIle or especially Ac-L-Ile. The results suggest that the equimolar mixture of Ac-L-Ile and Ac-D-aIle could be obtained from an Ac-L-Ile-excess mixture by recystallization from water.     

Functional Characterization of D-Galacturonic Acid Reductase,a Key Enzyme of the Ascorbate Biosynthesis Pathway,from Euglena gracilis

D-Galacturonic acid reductase, a key enzyme in ascorbate biosynthesis, was purified to homogeneity from Euglena gracilis. The enzyme was a monomer with a molecular mass of 38–39 kDa, as judged by SDS–PAGE and gel filtration. Apparently it utilized NADPH with a Km value of 62.5±4.5 μM and uronic acids, such as D-galacturonic acid (Km=3.79±0.5 mM) and D-glucuronic acid (Km=4.67±0.6 mM). It failed to catalyze the reverse reaction with L-galactonic acid and NADP⁺. The optimal pH for the reduction of D-galacturonic acid was 7.2. The enzyme was activated 45.6% by 0.1 mM H₂O₂, suggesting that enzyme activity is regulated by cellular redox status. No feedback regulation of the enzyme activity by L-galactono-1,4-lactone or ascorbate was observed. N-terminal amino acid sequence analysis revealed that the enzyme is closely related to the malate dehydrogenase families.     

Isolation of 6-hydroxy-L-tryptophan from the fruiting body of Lyophyllum decastes for use as a tyrosinase inhibitor

ABSTRACT

Tyrosinase is the key enzyme that controls melanin formation. We found that a hot water extract of the lyophilized fruiting body of the fungus Lyophyllum decastes inhibited tyrosinase from Agaricus bisporus. The extract was fractionated by ODS column chromatography, and an active compound was obtained by purification through successive preparative HPLC using an ODS and a HILIC column. Using spectroscopic data, the compound was identified to be an uncommon amino acid, 6-hydroxytryptophan. 6-Hydroxy-L-tryptophan and 6-hydroxy-D-tryptophan were prepared through a Fenton reaction from L-tryptophan and D-tryptophan, respectively. The active compound was determined to be 6-hydroxy-L-tryptophan by comparison of their circular dichroism spectra and retention time on HPLC analysis of the N^α-(5-fluoro-2,4-dinitrophenyl)-L-leuciamide derivative with those of 6-hydroxy-L-tryptophan and 6-hydroxy-D-tryptophan. A Lineweaver–Burk plot of the enzyme reaction in the presence of 6-hydroxy-L-tryptophan indicated that this compound was a competitive inhibitor. The IC₅₀ values of 6-hydroxy-L-tryptophan was 0.23 mM.     

Substrate Specificity of the Protease from Streptomyces cellulosae

The substrate specificity and the mode of action of the protease from Streptomyces cellulosae were investigated, using many kinds of peptides and proteins as substrates. The protease hydrolyzed peptides consisting of hydrophobic amino acids such as L-Phe-L-Leu-NH₂, L-Pro-L-Phe-NH₂, l-Leu-L-Met, L-Leu-L-Leu, Gly-L-Ile, L-Phe-L-Phe, L-Pro-L-Leu-Gly-NH₂, etc. The protease hydrolyzed zein best among the proteins tested, but weakly hydrolyzed gelatin, myoglobin, bovine serum albumin, γ-globulin, and collagen. The protease mainly hydrolyzed Ser¹²-Leu¹³, Leu¹³-Tyr¹⁴, and Tyr¹⁴-Gln¹⁵ bonds in the oxidized A-chain of insulin and at least the Leu¹⁵-Tyr¹⁶ bond in the oxidized B-chain of insulin.     

Gene Cloning of α-Methylserine Aldolase from Variovorax paradoxus and Purification and Characterization of the Recombinant Enzyme

The α-methylserine aldolase gene from Variovorax paradoxus strains AJ110406, NBRC15149, and NBRC15150 was cloned and expressed in Escherichia coli. Formaldehyde release activity from α-methyl-L-serine was detected in the cell-free extract of E.coli expressing the gene from three strains. The recombinant enzyme from V. paradoxus NBRC15150 was purified. The V _max and K _m of the enzyme for the formaldehyde release reaction from α-methyl-L-serine were 1.89 μmol min^?1 mg^?1 and 1.2 mM respectively. The enzyme was also capable of catalyzing the synthesis of α-methyl-L-serine and α-ethyl-L-serine from L-alanine and L-2-aminobutyric acid respectively, accompanied by hydroxymethyl transfer from formaldehyde. The purified enzyme also catalyzed alanine racemization. It contained 1 mole of pyridoxal 5′-phosphate per mol of the enzyme subunit, and exhibited a specific spectral peak at 429 nm. With L-alanine and L-2-aminobutyric acid as substrates, the specific peak, assumed to be a result of the formation of a quinonoid intermediate, increased at 498 nm and 500 nm respectively.     

N-Carbamyl-L-Amino Acid Amidohydrolase of Pseudomonas sp. Strain NS671: Purification and Some Properties of the Enzyme Expressed in Escherichia coli

An N-carbamyl-L-amino acid amidohydrolase was purified from cells of Escherichia coli in which the gene for N-carbamyl-L-amino acid amidohydrolase of Pseudomonas sp. strain NS671 was expressed. The purified enzyme was homogeneous by the criterion of SDS–polyacrvlamide gel electrophoresis. The results of gel filtration chromatography and SDS–polyacrylamide gel electrophoresis suggested that the enzyme was a dimeric protein with 45-kDa identical subunits. The enzyme required Mn²⁺ ion (above 1 mM) for the activity. The optimal pH and temperature were 7.5 and around 40°C, respectively, with N-carbamyl-L-methionine as the substrate. The enzyme activity was inhibited by ATP and was iost completely with p-chloromercuribenzoate (1 mM). The enzyme was strictly L-specific and showed a broad substrate specificity for N-carbamyl-L-α-amino acids.     

Production,Purification, and Characterization of D-Aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6

The best inducers for D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) were a poor substrate, N-acetyl-;-methyl-D-leucine, and an inhibitor, N-acetyl-D-alloisoleucine. The enzyme has been homogeneously purified. The molecular weight of the native enzyme was estimated to be 58,000 by gel filtration. A subunit molecular weight of 52,000 was measured by SD8–PAGE, indicating that the native protein is a monomer. The isoelectric point was 5.2. The enzyme was specific to the D-isomer and hydrolyzed N-acetyl derivatives of D-leucine, D-phenylalanine, D-norleucine, D-methionine, and D-valine, and also N-formyl, N-butyryl, and N-propionyl derivatives of D-leucine. The K_m for N-acetyl-D-leucine was 9.8mM. The optimum pH and temperature were 7.0 and 50°C, respectively. The stabilities of pH and temperature were 8.1 and 40°C. D-Aminoacylases from three species of the genus Alcaligenes differ in inducer and substrate specificities, but are similar with respect to molecular weight and N-terminal amino acid sequence.     

Microbial Conversion of DL-5-Substituted Hydantoins to the Corresponding L-Amino Acids by Pseudomonas sp. Strain NS671

A bacterial strain, NS671, which converts DL-5-(2-methylthioethyl)hydantoin stereospecifically to L-methionine, was isolated from soil and was classified into the genus Pseudomonas. With growing cells of Pseudomonas sp. strain NS671, DL-5-(2-methylthioethyl)hydantoin was effectively converted to L-methionine. Under adequate conditions, 34g of L-methionine per liter was produced with a molar yield of 93% from DL-5-(2-methylthioethyl)hydantoin added successively. In addition to L-methionine, other amino acids such as L-valine, L-leucine, L-isoleucine, and L-phenylalanine were also produced from the corresponding 5- substituted hydantoins, but these L-amino acids produced were partially consumed by strain NS671. The hydantoinase, by which 5-substituted hydantoin rings are opened, was ATP-dependent. The N-carbamylamino acid amidohydrolase was found to be strictly L-specific, and its activity was inhibited by high concentration of ATP.     

Effects of Calcium Ion on the Catalytic Activity of α-Chymotrypsin in Organic Solvents

Addition of small amounts of calcium ion markedly accelerated the transesterification of N-acetyl-l-tyrosine methyl ester to its ethyl ester by the catalysis of α-chymotrypsin in organic solvents. Maximum increase of the reaction rate was about 12-fold in the presence of 25 μm of calcium ion in ethanol. The rate increase was strongly dependent on calcium ion concentration and nature of organic solvents. Esterification of N-acetyl-l-tyrosine and hydrolysis of N-acetyl-l-tyrosine ethyl ester by α-chymotrypsin in organic solvents were also accelerated by calcium ion. The reactions obeyed Michaelis–Menten kinetics, and the acceleration of the reactions was due to the increase in k_cat.     

NADP-Specific Glutamate Dehydrogenase from Alkaliphilic Bacillus sp. KSM-635: Purification and Enzymatic Properties

An NADP-specific glutamate dehydrogenase [L-glutamate: NADP⁺ oxidoreductase (deaminating), EC 1.4.1.4] from alkaliphilic Bacillus sp. KSM-635 was purified 5840-fold to homogeneity by a several-step procedure involving Red-Toyopearl affinity chromatography. The native protein, with an isoelectric point of pH 4.87, had a molecular mass of approximately 315 kDa consisting of six identical summits each with a molecular mass of 52 kDa. The pH optima for the aminating and deaminating reactions were 7.5 and 8.5, respectively. The optimum temperature was around 60°C for both. The purified enzyme had a specific activity of 416units/mg protein for the aminating reaction, being over 20-fold greater than that for deaminating reaction, at the respective pH optima and at 30°C. The enzyme was specific for NADPH (K_m 44 μM), 2-oxoglutarate (K_m 3.13 mM), NADP⁺ (K_m 29 μM), and L-glutamate (K_m 6.06 mM). The K_m for NH₄Cl was 5.96 mM. The enzyme could be stored without appreciable loss of enzyme activity at 5°C for half a year in phosphate buffer (pH 7.0) containing 2 mM 2-mercaptoethanol, although the enzyme activity was abolished within 20 h by freezing at ?20°C.     

Depletion of L-arginine induces autophagy as a cytoprotective response to endoplasmic reticulum stress in human T lymphocytes

L-arginine (L-Arg) deficiency results in decreased T-cell proliferation and impaired T-cell function. Here we have found that L-Arg depletion inhibited expression of different membrane antigens, including CD247 (CD3ζ), and led to an ER stress response, as well as cell cycle arrest at G₀/G₁ in both human Jurkat and peripheral blood mitogen-activated T cells, without undergoing apoptosis. By genetic and biochemical approaches, we found that L-Arg depletion also induced autophagy. Deprivation of L-Arg induced EIF2S1 (eIF2α), MAPK8 (JNK), BCL2 (Bcl-2) phosphorylation, and displacement of BECN1 (Beclin 1) binding to BCL2, leading to autophagosome formation. Silencing of ERN1 (IRE1α) prevented the induction of autophagy as well as MAPK8 activation, BCL2 phosphorylation and XBP1 splicing, whereas led T lymphocytes to apoptosis under L-Arg starvation, suggesting that the ERN1-MAPK8 pathway plays a major role in the activation of autophagy following L-Arg depletion. Autophagy was required for survival of T lymphocytes in the absence of L-Arg, and resulted in a reversible process. Replenishment of L-Arg made T lymphocytes to regain the normal cell cycle profile and proliferate, whereas autophagy was inhibited. Inhibition of autophagy by ERN1, BECN1 and ATG7 silencing, or by pharmacological inhibitors, promoted cell death of T lymphocytes incubated in the absence of L-Arg. Our data indicate for the first time that depletion of L-Arg in T lymphocytes leads to a reversible response that preserves T lymphocytes through ER stress and autophagy, while remaining arrested at G₀/G₁. Our data also show that the L-Arg depletion-induced ER stress response could lead to apoptosis when autophagy is blocked.     

Detection of α-L-Arabinofuranosidase Activity in Isoelectric Focused Gels using 6-Bromo-2-naphthyl-α-L-arabinofuranoside

For the specific detection of α-L-arabinofuranosidase (α-L-AFase) activity in isoelectric focused gels, 6-bromo-2-naphthyl-α-L-arabino-furanoside (BN-α-L-Araf) was synthesized by the condensation of 2, 3, 5-tri-O-benzoyl-α-L-arabinofuranosyl bromide and 6-bromo-2-naphthol. α-L-AFase activity had been detected in a gel after isoelectric focusing by using the synthesized BN-α-L-Araf as a substrate, and the detection for the enzyme activity was more sensitive than protein detection with Coomassie Brilliant Blue R-250.     

Antioxidative Effect of an Isolate from a Culture Filtrate of Aspergillus niger

D-Ins(1,3,4,5)P₄ and unnatural L-Ins(1,3,4,5)P₄ were prepared in gram-quantities from D- and L-2,6-di-O-benzyl-myo-inositol by a chemical phosphorylation and deprotection step in high yield and purity without extensive purification. The optically pure benzyl derivatives were obtained by enzyme-catalyzed resolution of racemic 2,6-di-O-benzyl-myo-inositol under acyl-transfer conditions in vinyl acetate as the acyl donor. The lipase of Candida antarctica only acetylated regio- and enantio-selectively the L-enantiomer, providing exclusively L-5-acetyl-2,6-di-O-benzyl-myo-inositol, whereas the D-enantiomer remained unchanged.     

Effects of Methionine,Cystine and Taurine on Plasma Cholesterol Level in Rats Fed a High Cholesterol Diet

l-Methionine γ-lyase (EC 4.4.1.11) catalyzes α,β-elimination of l-2-amino-3-(N-methylamino)propionic acid and l-2-amino-3-(N-hydroxyethylamino)propionic acid to yield pyruvate, ammonia, and the corresponding amines. These amino acids also undergo the enzymatic β-replacement reaction with thiols to produce the corresponding S-substituted cysteines. Thus, l-methionine γ-lyase cleaves a C-N bond in addition to C-S, C-Se, and C-O bonds at the β position of amino acids by elimination and replacement reactions. A linear relationship between the reactivity, (log(V_max/Km) and the pKa value of the conjugated acid of the leaving group has been found for Se-methyl-l-selenocysteine, S-methyl-l-cysteine, and O-methyl-l-serine. However, l-2-amino-3-(N-methylamino)propionic acid has shown lower reactivity than that expected from the pKa value of methylammonium ions.     

Production of l-aspartic acid by biotransformation and recovery using reverse micelle and gas hydrate methods

l-Aspartic acid (l-Asp) was produced using Escherichia coli (ATCC 11303), and its recovery from the reaction mixture was studied using reverse micelle and gas hydrate methods. The effect of initial substrate concentration on l-Asp production was also investigated, and inhibition was shown to occur above 0.75 mol L^?1. The values of the kinetic constants were determined as r_max=2.33×10^?4 mol L^?1 min^?1, K_M=0.19 mol L^?1, and K_ss=3.98 mol L^?1. The reverse micelle phase used for extraction contained Aliquat-336, 1-decanol and isooctane, and a micro-injection technique was used for extraction of l-Asp. The reverse micelle system is a useful technique for obtaining small particle sizes, which can be used for the synthesis of nanoparticle biomolecules. Recovery of l-Asp from reverse micelles using CO₂ hydrates was carried out, giving a recovery of 55%. The formation of CO₂ hydrate from the reverse micelle solution breaks the micelle by reducing the amount of water in the micelle structure, thus precipitating the l-Asp.     

Purification and Characterization of UDP-Arabinopyranose Mutase from Chlamydomonas reinhardtii

Chlamydomonas reinhardtii cells are surrounded by a mixture of hydroxyprolin-rich glycoproteins consisting of L-arabinose, D-galactose, D-glucose, and D-mannose residues. The L-arabinose residue is thought to be attached by a transfer of UDP-L-arabinofuranose (UDP-Araf), which is produced from UDP-L-arabinopyranose (UDP-Arap) by UDP-arabinopyranose mutase (UAM). UAM was purified from the cytosol to determine the involvement of C. reinhardtii UAM (CrUAM) in glycoprotein synthesis. CrUAM was purified 94-fold to electrophoretic homogeneity by hydrophobic and size-exclusion chromatography. CrUAM catalyzed the reversible conversion between UDP-Arap and UDP-Araf and exhibited autoglycosylation activity when UDP-D-[¹⁴C]glucose was added as substrate. Compared to the properties of native and recombinant CrUAM overexpressed in Escherichia coli, native CrUAM showed a higher affinity for UDP-Arap than recombinant CrUAM did. This increased affinity for UDP-Arap might have been caused by post-translational modifications that occur in eukaryotes but not in prokaryotes.